1. Field of the Invention
This application relates generally to apparatus and methods for detecting and measuring analytes of interest by inducing electrochemiluminescence (ECL) in a test sample and detecting the resulting light.
Numerous methods and systems have been developed for detecting and quantitating analytes of interest in chemical, biochemical, biological, and environmental samples. Methods and systems that are capable of measuring toxins, environmental contaminants, pharmacological agents, bioactive substances, metabolites, pathogenic organisms, proteins and nucleic acids are of substantial value to researchers and clinicians. At this time, there are a number of commercially available instruments that utilize ECL for analytical measurements. These instruments have demonstrated exceptional performance.
The high cost, complex engineering and long development time required to custom-design and manufacture ECL instruments have delayed broad implementation of ECL technology. Clearly, there remains a need for ECL subsystems or modules that can be easily adapted to a broad variety of different applications.
Current needs for precision analytical testing instrumentation are extraordinarily diverse. For example, pharmaceutical screening analyses require instruments that can perform large numbers of analyses at very high speeds on very small quantities of sample. In addition, such instruments may need to perform many different types of highly sensitive quantitative tests utilizing different detection methods. Similarly, clinical diagnostic analyses for human health care typically require highly sensitive and exceptionally reliable instrumentation. In contrast, it is expected that commercial instruments intended for field use would be small, perhaps portable, simple to use, and operable with only limited power. Low production and maintenance costs are often predominant considerations.
2. Description of the Prior Art
An apparatus for carrying out electrochemiluminescence test measurements is found in U.S. Pat. No. 5,466,416 assigned to IGEN, Inc. A cross-sectional view of a flow cell is depicted in FIG. 1. Flow cell 18 comprises a removable plug 20, a gasket 22, a retainer block 24, a counter electrode 26, an ECL test chamber 28, a working electrode 30, a transparent block 32, a counter electrode 34, a retainer block 36, a conduit 46, a main housing 48, a chamber 40, a lateral block 42, a frit 44, a gasket 50, a plug 52, an O-ring seal 56, a threaded coupling 58, a conduit 60, a pivot arm 61, a magnet 62, and a threaded coupling 64.
Flow cell 18 includes a main housing 48 formed of a durable, transparent and chemically inert material such as acrylic or polymethyl methacrylate. Threaded coupling 64 defines a fluid inlet in a lower surface of housing 48 and is contiguous with conduit 46. Conduit 46 extends through housing 48 from coupling 64 to an upper surface of housing 48. Threaded coupling 58 defines a fluid outlet in a lower surface of housing 48 and is contiguous with conduit 60. Conduit 60 extends through housing 48 from coupling 58 to the upper surface of housing 48. ECL test chamber 28 is bounded by the upper surface of housing 48, a lower surface of block 32, lower and side surfaces of counter electrodes 26 and 34, the upper surface of working electrode 30, and the interior surface of gasket 22. Chamber 28 communicates with both conduit 60 and conduit 46. Fluid introduced through coupling 64 may travel through conduit 46 to chamber 28 and exit through conduit 60 and coupling 58.
Working electrode 30, counter electrode 26, and counter electrode 34 may consist of electrically-conductive materials such as platinum or gold. Working electrode 30 has a generally flat, elongate, rectangular shape having a longitudinal axis arranged generally transverse to a longitudinal axis of chamber 28. Electrode 30 is positioned centrally between conduits 60 and 46 in a shallow groove formed in the upper surface of housing 48. An adhesive (not shown) bonds electrode 30 to the groove in housing 48. Accordingly, at least three seams between electrode 30 and housing 48 abut chamber 28; one on each latitudinal side of electrode 30 and a third at a longitudinal end of electrode 30. As displayed in FIG. 1, electrode 30 is approximately as wide as the gap between counter electrodes 26 and 34 and is positioned centrally therebetween.
Counter electrodes 26 and 34 have an xe2x80x9cLxe2x80x9d-shaped cross-section, the shorter arm having a length slightly longer than the thickness of block 32 and the longer arm having a length of less than half of the width of block 32. The two arms of each electrode are flat, thin and positioned perpendicular to each other but in different planes. The widths of electrodes 26 and 34 are approximately less than half of the thickness of block 32. Counter electrode 26 is affixed to a side of transparent block 32 and is held in place by retainer block 24. On the opposite side of transparent block 32, counter electrode 34 is similarly affixed by retainer block 36.
Magnet 62 is affixed to pivot arm 61. In its raised position, pivot arm 61 positions magnet 62 beneath working electrode 30, sandwiching a segment of housing 48 therebetween. In its lowered position, pivot arm 61 pivots down and away from housing 48 thereby significantly increasing the distance between working electrode 30 and magnet 62.
A reference electrode assembly, integrated into housing 48, comprises chamber 40, block 42, gasket 50, frit 44, plug 52, and gasket 56. An ionic fluid (not shown) is retained within chamber 40. Chamber 40 comprises a cavity defined by housing 48, gasket 50 and block 42. Frit 44 extends into conduit 60 and is sealed by O-ring 56 and plug 52.to prevent fluidic interchange.
A refill aperture (not shown) is provided in housing 48 to allow replacement of the ionic fluid held in chamber 40. The refill aperture is sealed by removable plug 20. To achieve useful and reproducible ECL test measurements, flow cell 18 utilized a temperature-controlled environment. FIG. 2 illustrates an apparatus 80 from U.S. Pat. No. 5,466,416 for providing a temperature-controlled environment for flow cell 18. Apparatus 80 comprises a photomultiplier tube (PMT) 82, an insulating cover 92, a housing 94, a plurality of foil heaters 96, a circuit board 84, flow cell 18, a magnet 62, a pivot arm 61, a linear actuator 98, a coil spring 102, an air space 90, and a fan 104. For reference purposes, housing 48, block 42, retainer block 24, counter electrode 26, and block 32 are specifically labelled on flow cell 18.
Foil heaters 96 are positioned on the outer lateral surfaces and the outer lower surface of housing 94. The upper surface of housing 94 adjacent PMT 82 is formed of a transparent material while the remaining portions of housing 94 are preferable opaque. Insulating cover 92 covers foil heaters 96 as well as the remaining uncovered outer surfaces of housing 94 to provide thermal insulation and prevent the entry of light into flow cell 18. PMT 82 is a conventional photomultiplier tube mounted on the upper surface of housing 94. PMT 82 is physically large compared to the size of the flow cell, requires a high-voltage power supply, and is highly sensitive to the surrounding temperature and the presence of magnetic fields. It is preferable that PMT 82 be maintained at a relatively low temperature. Flow cell 18 is positioned below PMT 82 inside temperature-controlled housing 94.
Circuit board 84, incorporating operating electronics for apparatus 80, is mounted on an interior surface of housing 94 adjacent flow cell 18. As shown, linear actuator 98 is connected to coil spring 102 which, in turn, is connected to pivot arm 61. Magnet 62 is affixed to an end of pivot arm 61.
The temperature within housing 94 is controlled through the operation of foil heaters 96 in conjunction with fan 104. Fan 104, affixed to the interior surface of housing 94, circulates air within air space 90. Air space 90 extends throughout the interior of housing 94 and surrounds each component therein, including, specifically, flow cell 18. Air space 90 further includes an air gap between the upper surface of flow cell 18, e.g., block 32, and the upper interior surface of housing 94.
As described above, pivot arm 61, shown in its lowered position, can pivot upward to place magnet 62 within housing 48 of flow cell 18. Linear actuator 98, operating in conjunction with coil spring 102, causes pivot arm 61 to move.
In an ordinary operation, magnet 62 is raised into a position adjacent to working electrode 30 of flow cell 18 to attract magnetic particles in an assay fluid in chamber 28 to the vicinity of working electrode 30. Shortly thereafter, to avoid magnetic interference with the operation of PMT 82, magnet 62 is withdrawn from flow cell 18 prior to the induction of electrochemiluminescence in the assay sample fluid. Conventionally, magnet 62 is not positioned to collect magnetic particles during the application of electrical energy to the assay fluid. Magnet 62 is usually retracted before electrochemiluminescence is induced to avoid magnetic interference with ECL measurements by PMT 82. Removal of the magnetic field from working electrode 30 may allow a flowing-assay sample fluid to carry away magnetic particles collected there.
Methods of calibration for apparatus 80 convolve diagnosis of the effectiveness of bead capture and the effectiveness of the ECL cell. Therefore, calibration is preferably achieved using bead-based standards (e.g. magnetic beads coated with ECL labels).
As shown, apparatus 80 includes thermal insulation between PMT 82 and flow cell 18. PMT 82 is very temperature-sensitive in that heat increases the background noise signal generated by PMT 82. Typically, PMT 82 is maintained in a moderate to low temperature environment. Since the ECL process generates considerable heat, flow cell 18 is thermally isolated from PMT 82. The use of thermal insulating material between flow cell 18 and PMT 82 increases the length of the optical path from working electrode 30 to PMT 82 and, therefore, reduces the efficiency with which light emitted at working electrode 30 is transmitted to PMT 82.
Additionally, it should be readily apparent that the optical path between chamber 28 of flow cell 18 and PMT 82 includes multiple air-solid and solid-solid boundaries. These transitions between media reduce the amount of ECL-generated light which ultimately reaches PMT 82. Light generated between counter electrode 26 and working electrode 30 or between counter electrode 34 and working electrode 30 passes from the assay fluid in chamber 28 through a bottom surface of block 32, through the bulk of block 32 and through the upper surface of block 32. At the lower surface of block 32, light is reflected back towards housing 48 and, in particular, working electrode 30. Light travelling through the bulk of block 32 is diffused and may be gradually separated into component wavelengths. At the upper surface of block 32, a portion of the incident light is internally reflected back into the bulk of block 32 while the remainder is transmitted into air space 90. Additionally, at the boundary between block 32 and air space 90, the light rays will be bent away from PMT 82 due to the decrease in refractive index across the boundary. Consequently, the amount of light directed towards PMT 82 is reduced.
The light travels through air space 90 to the lower surface of housing 94 where, again, some light is reflected back towards flow cell 18 while the remainder is transmitted into the bulk of housing 94. Within the bulk of housing 94, the light is diffused and may be further caused to separate into component wavelengths. At the upper surface of housing 94, where PMT 82 abuts housing 94, a portion of the light is internally reflected into the bulk of housing 94 while a remainder portion is transmitted to PMT 82. The aforedescribed diffusion, bending, and reflection of light may significantly reduce the amount of ECL-generated light which is actually incident upon PMT 82.
As shown, flow cell 18 includes electrode-housing seams within ECL chamber 28. The adhesive present at these seams and used to affix working electrode 30 to housing 48 may deteriorate and erode over time. As a result, assay fluid components, cleaning fluid components, or other materials may collect in the seams between electrode 30 and housing 48. The collected materials may react with or otherwise contaminate components of subsequent assays and thereby affect assay results.
It is, therefore, a primary object of the present invention to provide apparatus and methodology for carrying out improved electrochemiluminescence test measurements.
A further object of the invention is to provide apparatus and methodology for the efficient detection of light generated during an electrochemiluminescence assay.
Still a further and related object of the invention is to provide a modular ECL measurement apparatus for rapid and efficient incorporation into an application-specific diagnostic device.
Another object of the invention is to provide apparatus and methodology for conducting electrochemiluminescence test measurements under conditions of continuous fluid flow upon an assay sample containing magnetic particles.
A still further object of the invention is to provide apparatus and methodology for applying a magnetic field to assay materials during the induction of electrochemiluminescence and simultaneously detecting the light generated thereby.
Another object of the invention is to provide apparatus that integrates each of the components needed to perform an ECL measurement in a single open-architecture ECL module.
Yet another object of the invention is to provide a modular apparatus for carrying out an ECL measurement that comprises a modular system interface.
A further object of the invention is to provide apparatus and methodology for an integrated system for assaying one or more samples for one or more analytes of interest.
A related object of the invention is to provide apparatus for conducting multiple simultaneous or near-simultaneous ECL measurements and for sharing an assay sample sampling device, a power supply, a controller, a system interface, and a user interface.
An additional object of the invention is to provide apparatus and methodology for normalizing the operations of two or more ECL modules.
Another object of the invention is to provide an apparatus for ECL measurements that comprises a modular system interface that is adapted for convenient coupling to other analytical or processing devices.
Another object of the invention is to provide apparatus and systems capable of detecting analytes in a sample by means of electrochemiluminescence and one or more other analytical techniques.
Still another object of the invention is to provide an integrated system for processing samples, amplifying nucleic acids, and measuring nucleic acids.
These and other objects of the invention are achieved in an apparatus for the conduct of electrochemiluminescence measurements which includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode abutting the ECL chamber and in optical registration with the transparent portion, a counter electrode abutting the ECL chamber and an electrically-shielded window adjacent to and in optical registration with the transparent portion of the cell wall.
The apparatus of the invention may also include a photodetector, e.g. a photodiode, in optical registration with the electrically-shielded window, the transparent portion of the cell wall and the working electrode.
In preferred embodiments of the invention, the working electrode is removably fitted within the cell and has a planar electrode surface abutting the ECL chamber such that no seam is created between the working electrode and the ECL chamber. A removable magnet is provided for applying a magnetic field to the working electrode.
The object of creating an integrated system for assaying a sample or plurality of samples for a plurality of analytes of interest is also achieved in systems comprising a plurality of modules which may share a common sample handling subsystem, a common power supply, a common controller and/or a common system or user interface.
According to an aspect of the present invention an apparatus for the conduct of electrochemiluminescence measurements includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode abutting the ECL chamber and in optical registration with the transparent portion, a counter electrode abutting the ECL chamber, and an electrically-shielded window adjacent to and in optical registration with the transparent portion.
According to another aspect of the present invention an apparatus for the conduct of electrochemiluminescence measurements includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode abutting the ECL chamber and in optical registration with the transparent portion, a counter electrode abutting the ECL chamber, a photodiode in optical registration with the transparent portion, and an optical filter adjacent to and in optical registration with the transparent portion.
According to another aspect of the present invention an apparatus for the conduct of electrochemiluminescence measurements includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode abutting the ECL chamber and in optical registration with the transparent portion, and a counter electrode abutting the ECL chamber and having an aperture in optical registration with the transparent portion.
According to still another aspect of the present invention an apparatus for the conduct of electrochemiluminescence measurements includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode abutting the ECL chamber and in optical registration with the transparent portion, and a counter electrode abutting the ECL chamber, wherein the working electrode is removably fitted within the cell and has a planar electrode surface abutting the ECL chamber.
According to still another aspect of the present invention an apparatus for the conduct of electrochemiluminescence measurements includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode having a planar electrode surface abutting the ECL chamber and in optical registration with the transparent portion of the cell wall, the working electrode being positioned within the cell such that no seam between the working electrode and the cell abuts the ECL chamber, and a counter electrode abutting the ECL chamber.
According to still another aspect of the present invention an apparatus for the conduct of electrochemiluminescence measurements includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode abutting the ECL chamber and in optical registration with the transparent portion, a counter electrode abutting the ECL chamber, a photodiode adjacent to and in optical registration with the transparent portion, and a magnetic field generating device operable to apply a magnetic field at the working electrode.
According to yet another aspect of the present invention an apparatus for the conduct of electrochemiluminescence measurements includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode abutting the ECL chamber and in optical registration with the transparent portion, a counter electrode abutting the ECL chamber, and a photodiode adjacent to and in optical registration with the transparent portion, the photodiode having a detection sensitivity substantially limited to light having a wavelength in a range of 400 nm to 900 nm.
According to yet another aspect of the present invention an apparatus for the conduct of electrochemiluminescence measurements includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode abutting the ECL chamber and in optical registration with the transparent portion, a counter electrode abutting the ECL chamber and having an aperture in optical registration with the transparent portion, a photodetector adjacent to and in optical registration with the transparent portion, and a magnetic field generating device, in registration with the aperture, operable to apply a magnetic field to the working electrode.
According to another aspect of the present invention an apparatus for the conduct of electrochemiluminescence measurements includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode abutting the ECL chamber and in optical registration with the transparent portion, a counter electrode abutting the ECL chamber, a photodiode adjacent to and in optical registration with the transparent portion, a magnetic field generating device operable to apply a magnetic field to the working electrode, and a magnetic field detector, in registration with the magnet device.
According to another aspect of the present invention an apparatus for the conduct of electrochemiluminescence measurements includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode abutting the ECL chamber and in optical registration with the transparent portion, a counter electrode abutting the ECL chamber, a photodiode, adjacent to and in optical registration with the transparent portion, for detecting electrochemiluminescence induced in an assay fluid in the ECL chamber and for producing an ECL signal representative of an intensity of the electrochemiluminescence, a storage device, coupled to the photodiode, in which a calibration signal representative of a calibration electrochemiluminescence may be stored, and a processor, coupled to the photodiode and to the storage device, operable to calculate an intensity value as a function of the ECL signal and the calibration signal.
According to another aspect of the present invention a cell for the conduct of electrochemiluminescence measurements includes a first base having a first interior surface, a planar working electrode positioned on the first interior surface, a second base having a second interior surface and having a transparent portion therein to allow light to pass therethrough, a planar counter electrode positioned on the second interior surface, the counter electrode having at least one opening therein to allow the light to pass therethrough in registration with the working electrode and the transparent portion of the second base, a gasket positioned between the working electrode and the counter electrode to define therebetween a cell volume, the volume communicating with the opening in the counter electrode, and a retaining device, coupled to the bases, wherein the interior surfaces of the bases are in opposing relationship to form the cell and wherein the second base includes a conduit through which fluid may be introduced into and removed from the cell volume.
According to another aspect of the present invention a cell for the conduct of electrochemiluminescence includes cell structural elements, a working electrode and a counter electrode, at least one of the structural elements having a transparent portion therein, wherein the working electrode is mounted on an interior surface of a structural element, a portion. of the working electrode and the transparent portion of the at least one structural element defining, at least in part, a chamber for the conduct of electrochemiluminescence, the working electrode including the entirety of a continuous planar surface of the chamber and the portion of the working electrode and the transparent portion of the structural element being optically in registration with one another.
According to another aspect of the present invention a method for conducting an ECL measurement includes the steps of introducing an assay sample into an ECL chamber within a flow cell, simultaneously applying an electric field and a magnetic field to the assay sample in the ECL chamber, and measuring, through an electrically-shielded window defining a wall of said ECL chamber, electrochemiluminescence induced in the assay fluid in the ECL chamber while the electric field and the magnetic field are applied.
According to another aspect of the present invention a method for conducting an ECL measurement includes the steps of introducing an assay sample into an ECL chamber within a flow cell, simultaneously applying an electric field and a magnetic field to the assay sample in the ECL chamber, and measuring with a semiconductor photodetector electrochemiluminescence induced in the assay fluid in the ECL chamber while the electric field and the magnetic field are applied.
According to another aspect of the present invention a method for normalizing a plurality of ECL measurement instruments includes the steps of conducting an ECL measurement with a reference ECL measurement instrument upon a reference sample to produce a reference ECL signal, conducting an ECL measurement with a test ECL measurement instrument upon the reference sample to produce a test ECL signal, and calculating a correction transform function as a function of the reference ECL signal and the test ECL signal.
According to another aspect of the present invention an apparatus for the conduct of assay measurements includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode abutting the ECL chamber and in optical registration with the transparent portion, a counter electrode abutting the ECL chamber, a first light detector, optically coupled to the ECL chamber and in optical registration with the transparent portion, for detecting electrochemiluminescence induced within the ECL chamber, a light source, optically coupled to the ECL chamber, for providing light to the ECL chamber, and a second light detector, optically coupled to the ECL chamber.
According to another aspect of the present invention an assay system includes a plurality of ECL modules and a controller device coupled to each of the plurality of ECL modules and operable to control an operation of each of the plurality of ECL modules.
According to another aspect of the present invention an assay system includes a plurality of ECL modules and a power supply coupled to each of the plurality of ECL modules and operable to supply electrical power to each of the plurality of ECL modules.
According to another aspect of the present invention an assay system includes a plurality of ECL modules and a sample introduction device coupled to each of the plurality of ECL modules and operable to supply a sample to each of the plurality of ECL modules.
According to another aspect of the present invention an assay system includes a plurality of ECL modules and a waste handling device coupled to each of the plurality of ECL modules and operable to receive waste from each of the plurality of ECL modules.
According to another aspect of the present invention an assay system includes a temperature-controlled enclosure and a plurality of ECL modules positioned within the temperature-controlled enclosure.
According to another aspect of the present invention an assay system includes an ECL module having an assay fluid outlet and an assay module having an assay fluid inlet coupled to the assay fluid outlet.
According to another aspect of the present invention an assay system includes an assay module having an assay fluid outlet and an ECL module having an assay fluid inlet coupled to the assay fluid outlet.
According to another aspect of the present invention an assay system includes an ECL module having a first assay fluid inlet and a first waste fluid outlet and an assay module having a second assay fluid inlet coupled to first assay fluid inlet and having a second waste fluid outlet coupled to the first waste fluid outlet.
According to another aspect of the present invention a modular ECL assay subsystem adapted for connection to and use with a power supply, a controller, and a fluid exchange system common to a plurality of the modular ECL subsystems includes a cell having at least one cell wall which includes a transparent portion adjacent to an ECL chamber defined within the cell, a working electrode abutting the ECL chamber and in optical registration with the transparent portion, a counter electrode abutting the ECL chamber, a light detector, optically coupled to the ECL chamber, for detecting electrochemiluminescence induced within the ECL chamber, a waveform generator coupled to at least one of the working electrode and the counter electrode and operable to generate an electric signal, a subsystem controller coupled to the waveform generator and operable to control an operation of the waveform generator, and an interface to the cell, coupled to each of the subsystem controllers, to the power supply, to the controller, and to the fluid exchange system, the controller being operable to control the subsystem controller, the power supply being operable to supply electrical power to the subsystem controller and the fluid exchange system being operable to provide an assay fluid to the cell and to receive a waste fluid from the cell.